Nucleic Acids
NMR Spectroscopy
Markus W. Germann
Departments of Chemistry and Biology
Georgia State University
2014!
NMR of Nucleic Acids 1
!
!
1)  Primary Structure of DNA and RNA!
2)  Resonance Assignment of DNA/RNA by Homonuclear NMR!
A)
B)
C)
D)
E)
1H
Chemical shifts !
Assignment of exchangeable protons!
Assignment of non-exchangeable proton!
Typical NOEs in helical structures!
Correlation between non-exchangeable !
!and exchangeable protons!
NMR Spectroscopy is an Important Method for Structural Studies of
Nucleic Acids:
PDB Holding, March 21, 2012!
Molecule!
Technique!
Proteins!
X-ray Diffraction!
Protein/Nucleic !
Acid Complexes!
Other!
65’703!
1’266!
3’331!
-!
70’302!
8’163!
933!
228!
-!
9’331!
430!
24!
122!
-!
492!
74’294!
2’223!
3’681!
-!
80’264!
NMR!
Other 1)!
total!
Nucleic Acids!
1) EM, Hybrid, other!
http://www.rcsb.org/pdb
Alternate Bases & Modifications (small selection):
HETERO BASE PAIRS
H
H3 C
N
N
H
O
H
N
N
N
H
N
N
N
H
+
N
N
O
H
H
N
N
H
N
H
N
N
O
O
Hoogsteen
H
H
H 3C
O
H
N
N
H
O
N
N
N
N
N
H
N
N
N
O
O
H
H
N
N
H
O
H
H
N
N
N
H3 C
O
H
O
N
N
N
N
N
Watson-Crick
O
N
N
H
N
N
N
N
H
Reverse Watson-Crick
N
N
N
HH N
H
Germann et al., Methods in Enzymology
(1995), 261, 207-225.
Nucleic acids: structures, properties, and
functions (2000) By Victor A. Bloomfield,
Donald M. Crothers, Ignacio Tinoco
HOMO BASE PAIRS
H
O
H
N
H
N
N
+
N
N
N
H
H
O
CC +
O
O
CH3
N
N
CH3
H
H
O
CH3
N
O
N
N
O
H
N
O
N
H
N
O
TT(I)
H3 C
H
N
N
N
N
N
N
N
H
N
H
N
N
O
N
N
O
N
H
N
TT(II)
NH 2
N
H
N
O
N
H
NH 2
AA(II)
N
H
N
H
N
H
N
H
H
N
N
H
N
O
N
H
N
N
N
N
H
N
N
N
N
N
GG(II)
N
N
AA(I)
N
N
O
H
H
GG(I)
Germann et al., Methods in Enzymology
(1995), 261, 207-225.
Nucleic acids: structures, properties, and
functions (2000) By Victor A. Bloomfield,
Donald M. Crothers, Ignacio Tinoco
Structure Determination:
I)
Assignment
II)
Local Analysis
•glycosidic torsion angle, sugar puckering, backbone conformation
base pairing
Global Analysis
•sequential, inter strand/cross strand, dipolar coupling
III)
Nucleic Acids have few protons…..
•NOE accuracy
> account for spin diffusion
•Backbone may be difficult to fully characterize
> especially α and ζ.
•Dipolar couplings
Chemical shift ranges in nucleic acids!
NH (G, T, U)!
NH2 (G, C, A)!
!
A-T!
G-C!
A-U!
!
Loops, MM!
Loops, MM!
H2, H8, H6!
H2, H8, H6!
H1’! H3’! H4’,H5’,H5’’! H2’,H2’’!
H1’! H2’,H3’,H4’,H5’,H5’’!
DNA!
RNA!
G-C!
15!
10!
5!
0!
RNA!
DNA!
H1'
H2'
H2''
H3'
H4'
H5'
H5''
5-6!
H1'
2.3-2.9(A,G) 1.7-2.3(T,C)! H2'
2.4-3.1(A,G) 2.1-2.7(T,C)! H3'
4.4-5.2!
H4'
3.8-4.3!
H5'
3.8-4.3!
H5''
3.8-4.3!
C1'
C2'
C3'
C4'
C5'
83-89!
35-38!
70-78!
82-86!
63-68!
C1'
C2'
C3'
C4'
C5'
5-6!
4.4-5.0
4.4-5.2!
3.8-4.3!
3.8-4.3!
3.8-4.3!
87-94!
70-78!
70-78!
82-86!
63-68!
9R-Borano DNA•RNA!
5’-d(A T G G T G C T C)!
(u a c c a c g a g)r-5’!
Adenine
!
!
!Guanine!
!
H2
H8
N6H
-
!C2
!C8
!N6
!152-156
!137-142
!82-84
!!149-151
!119-121
!157-158
!214-216
!220-226
!224-232
!166-172
!!H8
!N1H
!N2H
!
!
!
!
!7.5-8
!7.7-8.5
!5-6/7-8
!!
!
!
!
!
!
!C4
!C5
!C6
!N1
!N3
!N7
!N9
!
!!7.5-8.3
!12-13.6
!5-6/8-9
!
!
!
!
!
!
!
!C2
!C8
!N1
!N2
!C4
!C5
!C6
!N1
!N3
!N7
!N9
!156!
!131-138!
!146-149!
!72-76 !
!152-154 !
!117-119 !
!161 !
!146-149!
!167 !
!228-238 !
!166-172!
!
Thymidine! !
!
!!
!Uridine
H6
Me5
N3H
-
!H6 !6.9-7.9 !C6
!H5 !5.0-6.0 !C5
!N3H!13-14 !N3
!- !- !
!!
!
!
!C2
!
!
!
!C4
!
!
!
!C5
!
!
!
!N1
!6.9-7.9!C6 !137-142
!1.0-1.9!Me5 !15-20
!13-14 !N3 !156
!!- !!
!C2 154
!
!C4 169
!
!C5 !95-112
!
!N1 !144
!
!
!Cytidine!
!137-142
!102-107
!156-162
!
!154
!169
!102-107
!142-146
!H6 !6.9-7.9
!C6 !136-144!
!H5 !5.0-6.0
!C5 !94-99!
!- !!N3 !210
!!
!N4H!6.7-7/81-8.8!N4 !94-98 !
!
!
!C2 !159!
!
!
!C4 !166-168!
!
!
!C5 !94-99!
!
!
!N1 !150-156!
No Structure Required!
Often, depending on the question asked, a full structure
determination is not required
! Does it form a duplex?
! Which base pairs are thermo labile?
! Which base pair is which… assignment?
! Is the loop structured?
! Structure
DNA Hairpin
Thermal lability!
Germann et al., Nucl. Acids Res. 1990 18: 1489-1498
AT!
GC!
T!
imino 1H NMR
31P
“New” DNAG9constructs
G3
!
O
5´-5´
T6
! Do the duplexes form, is there alpha
base pairing?
G
!
!
α
O
O
! Does the unusualG1base pair form?
!
!!
CH 3
!
O
!
O
alpha C
G9
T6
alpha T
G1
C1!
!
O
β
G3
N
!
G3
!
alpha A
O
T6
!!
CH 3
O
!
G9/G1
T7
!
G3
G1
T7
NH
!
HN
G9
N
O
T6 T7
C1!
3´-3´
!
T7
NMRO!
5’-G
C
C
G
G A A
C α!T! T
T α!T! C
A A G
G
C
C!
G-5’!
5’-G
C
C
G
G
C
T
A
G
C
C!
G-5’!
G9/G1
T7 T6
G3
control
14.0
13.5
13.0
12.5
ppm
1.0
0.5
0.0
A
T
A
T
-0.5
-1.0
T
A
C
G
-1.5
ppm
WNV-RNA
RNA 1! 7bp!
G48!
G50!
RNA 4!
U49!
RNA 2! 11bp!
U20!
U64!
!
5bp
G54!
G62! G26!
G18!
RNA 4!
GAGA!
U22! G61!
G! G!
U28!
!
A
A
40! A
A
A
C G
C G
A U
RNA 1!
A
G
C G
A U
C G 50!
G C
G
30!U A
C G
U A
C
A
G C
U A
C
C
A
U A 60!
G 10!
U
A
U G
A
C G
C
G
20!U A
G
U!
A U
G! A
G C
G
U
G A U
C
3’!
C
RNA 4!
RNA 3!
!
+ 4U!
RNA 2!
5’!
G!
RNA 3! 19bp!
RNA 5; RNA 6 G10->C!
RNA 5!
Fibrinogen Specific DNA Aptamer
A
288K
B
308K
Figure 2: 12% Native PAGE to observe mobilities of Ap90. Ap90 is
compared to single stranded oligomers of various lengths. The lanes
were loaded from the smallest to the largest sequences with Lane A-E
containing the 10-mer, 18-mer, 30-mer, 50-mer, and the 90-mer
respectively. Lane F contains the aptamer Ap90. The smear in lane F
encompassing a large range of DNA sizes (~90 nucleotides - ~30
nucleotides) indicates that the aptamer has multiple conformations.!
Hamilton & Germann 2011
Solvent Suppression
The presence of an intense solvent resonance necessitates an impractical high
dynamic range. 110 M vs <1mM (down to 5-10 uM)
To overcome this problem several methods are currently applied:
1) Presaturation 2) Observing the FID when the water passes a null condition after a 180 degree
pulse.
3) Suppression of broad lined based on their T2 behavior. 4) Selectively excitation, with and without gradients
5a) Use of GRASP to select specific coherences thereby excluding the intense
solvent signal. In this case the solvent signal never reaches the ADC. This
allows the observation of resonances that are buried under the solvent peak.
5b) Use of GRASP to selectively dephase the solvent resonance (WATERGATE)
5c) Excitation sculpting
!
Presat
Selective Excitation
90
90
P18
P18
x
180
y
td
td
SINGLE LINE ON RESONANCE
Presaturation field strength: 20-40 Hz corresponds to a 6-12ms 90deg pulse
Pros:
Easy to set up
Excellent water suppression
Cons:
Resonances under water signal!
(T variation)
Labile protons not visible
(some GC pairs may be)
!
Bo
Bz
My
M =0
Selective rf pulse on solvent resonance followed by a gradient
pulse to dephase the waterBsignal.
z
This could be followed by a mild presaturation field. The
selective rf pulse (1-2ms, depending on width to be zeroed) is
usually of the gauss type.
The selective rf pulse z-gradient constructs could be repeated
(WET).
!
Jump and return
90
-x
90
x
d1
Watergate
td
z
90
x
180
x
90
-x
90
-x
Δ
Δ
z
y
y
x
Δ
y
Δ
x
solvent
G
z
+1
+1
p 1G 1 + p2G 2 ...... = 0
Pros:
Easy to set up
Excellent water suppression
(with proper setup as good as presat)
Good for broad signals!
Cons:
Non uniform excitation Baseline not flat
Other sequences: 1331 etc
!
Pros:
Cons:
!
Water!
Excellent water suppression
Uniform excitation
Baseline flat
May loose broad resonances
Exitation Sculpting
T.-L. Hwang & A.J. Shaka, J. Mag. Res. (1995), 112 275-279!
1D!
Pros:
Easy to set up
Excellent water suppression
“ok” for broad signals!
Uniform excitation
Cons:
May loose some intensity on very broad signals
Spectra: 1.5mM DNA in Water, Nanjunda, Wilson and Germann unpublished!
NOESY!
Interesting structures have often broad imino protons.
àMost modern techniques obliterate them.
A"
B"
Jump and return
!7˚$C$
to the rescue
ppm
5.0
4.5
4.0
3.5
ppm
3.0
+ supercooled conditions
3.5
5'-
G
C
T
A
C
G
C αA
G T
G
C
G
C
A
T
C
G
G
C
-5'
4.0
4.5
5.0
!8˚$C$
ppm
+17˚$C$
+7˚$C$
3.5
4.0
4.5
+17˚$C$
5.0
Spring, A.M. & Germann, M.W., Anal. Biochem., 2012.!
14.0 13.8 13.6 13.4 13.2 13.0 12.8 12.6 12.4
ppm
1
2
3
4
6
7
8
9 10
! !
5’-G
C
T
A
C
G
C α!A! G
G T C
G
C
A
T
C
G
5
G!
C-5’!
20 19 18 17 16 15 14 13 12 11!
αA5 H2!
C4 H5!
ppm!
6.0!
6.5!
T2!T
13!
G12!
G1! G6!
G7!
G17!
G
18!
G10!
C 4 A N!
7.0!
αA5 H2!
7.5!
8.0!
T16!
C 4 A B!
8.5!
G17Im!
Structure Determination, NMR experiments:
I)
Assignment
II)
Local Analysis
•glycosidic torsion angle
•sugar puckering
•backbone conformation
•base pairing
(NOE, COSY)
(COSY, COSY, NOE, +)
(COSY, +)
(NOE, COSY)
Global Analysis
•sequential
•inter strand/cross strand
•dipolar coupling
(NOE, COSY)
(NOE, COSY)
(HSQC, HSQC)
III)
NOESY, COSY, HSQC
TOCSY……
Black: unlabeled, Blue: labeled DNA or RNA
Stereospecific Assignment
O!
H5!'!!
H5!'!'!
B!
O!
H3!'!!
H2!'!!
H4!'!!
H2!'!'!
O!
H1!'!!
2’’!
Deoxyribose!
O!
2’!
H2ʼʼ
H2ʼ
H5ʼ
H5ʼʼ
H5!'!!
H5!'!'!
H3!'!!
B!
O!
H2!'!!
H4!'!!
How do we determine them?
H1!'!!
O!
OH!
Ribose!
0 ppm
a) Rule of Thumb (5’ downfield of 5’’)
Shugar and Remin BBRC (1972), 48, 636-642)
b) Short mixing times NOESY
dH1’H2” shorter than H1’H2’
-> Crosspeak H1’-H2’’ > H1’H2’
0 ppm
Structure Determination:
I)
Assignment
II)
Local Analysis
•glycosidic torsion angle, sugar puckering,backbone conformation
base pairing
Global Analysis
•sequential, inter strand/cross strand, dipolar coupling
III)
Nucleic Acids have few protons…..
•NOE accuracy
> account for spin diffusion
•Backbone may be difficult to fully characterize
> especially α and ζ.
•Dipolar couplings
Distance information determines the glycosidic torsion angle
3.8Å!
2.5Å!
! How do we get distance information?
o Nuclear Overhauser effect (< 6Å)
Distance information determines the glycosidic torsion angle
3.8Å!
2.5Å!
! How do we get distance information?
o Nuclear Overhauser effect (< 6Å)
Sugar puckering!
The five membered furanose ring is not planar. It can be puckered in an envelope
form (E) with 4 atoms in a plane or it can be in a twist form. The geometry is
defined by two parameters: the pseudorotation phase angle (P) and the
pucker amplitude (Φ).
In general:
RNA (A type double helix) C3' endo.
DNA (B type double helix) C2' endo.
=
m
cos (P + 144 (j-2))
m
range: 34° - 42°
O3’
2’endo!
α
=
3+
125°
nucleotide unit
j
3’endo!
β
γ
ν4
ν3
δ
ε
ν2
O4’
χ
ν0
ν1
N (Northern)!
5’!
3’endo!
(Angle ~ 90 deg)!
5’!
2’endo!
S!
(Angle ~ 170 deg)!
(Southern)!
2’endo sugar H1’, H2’, H2”, H3’ region!
H1’!
H3’!
H2”!
H2’!
Widmer,H. and Wüthrich,K. (1987) J. Magn. Reson.,
74, 316-336.
3’endo sugar H1’, H2’, H2”, H3’ region!
H1’!
H3’!
H2”!
H2’!
Sugar puckering!
H1’!
H1’!
H3’!
H3’!
H2’!
H2’!
H2”!
H2”!
3’endo sugar!
2’endo sugar!
H1’!
LFA- COSY!
2’endo sugar H1’, H2’, H2” region!
H2’’!
H2’!
90
45
t1
Sugar puckering!
C3´-endo C2´-exo: P = 0°!
J1’2’ 2 Hz (with P = 9) N!
C2´-endo: P = 162°!
J1’2’ 9 Hz (with P = 144) S!
In a 50/50 situation the measured J 1’2’ is 5.5 Hz which would!
correspond to P of 70 degree.!
Sugar puckering!
Usually (DNA) one observes equilibrium of the S and N forms sugar repuckering. Unless one form greatly dominates the local analysis requires quite a
few parameters: PN , PS , ΦN , ΦS , fS
Several methods for analysis exist, graphical and the more rigorous simulation.
In practice the desired outcome determines the effort to be made. Sums of the
coupling constants are often easier to obtain.
fS = (Σ 1’ –9.8)/5.9
Σ 1’ = J 1’2’ + J 1’2’’
Σ 2’ = J 1’2’ + J 2’3’ + J 2’2’’
Σ 2’’ = J 1’2’’ + J 2’’3’ + J 2’2’’
Σ 3’ = J 2’3’ + J 2’’3’ + J 3’4’
See also our pure examples:
fS=0 and ~1 respectively
If fs < 50% J1’ 2’ < J1’ 2’’
If fs ca 0% J1’ 2’ very small
If fs > 70% J1’ 2’ > J1’ 2’’
Sugar puckering!
Nt
control
Σ1´
fs
alphaT
Σ1´
fs
alphaC
Σ1´
fs
alphaT!
alphaA
alphaG
5’-G
Σ1´ C fs G AΣ1´ A Tfs α!T! C
C G C α!T! T A A G
0.81 14.3 0.76
14.9 0.86
G1
15.2
0.92
15.3
0.93
14.6
C2
15.1
0.90
14.7
0.83
15.0
0.88
15.0
0.88
0.86
16.0
1.00
9.4
-
300
T6
G
C
C!
G-5’!
(15.3) (0.93)
30
G3
16.2
1.00
15.9
1.00
14.9
A4
16.2
1.00
15.3
0.93
15.3
0.93
100
10.7
-
14.5
0.80
A5
15.7
1.00
15.3
0.93
15.1
0
0.90
12.1
0.39
14.9
0.86
T6
15.1
0.90
15.3
0.93
14.7
300
0.83
αT7
14.6
0.81
14.8
0.84
T7
16.0
1.00
12.3
-
200
(15.3)
P (0.93)
15.0
0.88
15.6
0.98
20
C8
15.1
0.90
12.9
0.53
9.5
G9
15.7
1.00
14.7
0.83
13.5
C10
(14)
(0.7)
(14)
(0.7)
(14)
200
100
20
10
0
30
-
15.0
0.88
14.4
0.82
10
0.63
14.3
0.76
14.9
0.86
0
30
(14)
(0.7)
(14)
(0.7)
20
0
300
C8
(0.7)
200
100
10
0
0
40
80
Time (ps)
Aramini, 2000, J. Biomolecular NMR, 18, 287-302
%
120
160
0
120
240
0
P
MD calculation!
MD-Tar calculation!
Pseurot calculations!
ΦS,N
!= 37°!
PS
!= 125-165 !
fS
!
!= 0.44-0.55!
ΦS,N
!= 37°!
PS
!= 130-155!
fS
!
!= 0.78-0.86!
!
!
!
!
Aramini, et al., 1998, Nucleic Acid Research, 26, 5644-5654
van Wijk,J., Haasnoot,K., de Leeuw,F.,
Huckreide,D. and Altona,C. (1995) PSEUROT 6.2.
A Program for the Conformational Analysis of Five
Membered Rings. University of Leiden, The
Netherlands
Introduction to Cross-Correlated Relaxation
Relaxation in NMR
à determines experimental strategies and experiments
à dynamic and structural parameters
Mechanisms
à Dipole -dipole
à CSA (e.g. 31P at higher fields; proportional to B2)
à Scalar relaxation (first and second kind)
à paramagnetic, etc
Recently it became possible to use cross correlated relaxation (CCR) to directly
measure bond angles without using a calibration curve as is needed for J’s.
à DD -DD
à DD -CSA
θ
Sugar Puckering from Cross-Correlated Relaxation
Γ DD-DD
ΓC1’H1’-C2’H2’ = k (3cos2θ-1)τc
θ = 180:
for 2’endo (B form)
Large and positive
θ = 90:
for 3’endo (A form)
Small and negative
Pseudorotation Phase Angle
θ1’2’ = 121.4°+1.03 ψm cos(P-144°)
BioNMR in Drug Research (2003) Chapter 7 p147-178. Christian Griesinger
Sugar puckering: Summary!
à Coupling constants: COSY, E.COSY, low flip angle COSY!
!Homonuclear, Heteronuclear!
à CT NOESY!
à CSA-DD and DD-DD cross correlated data!
à 13C chemical shifts, in favorable cases!
Some references!
!
Szyperski, T., et al. (1998). JACS. 120, 821- 822.
Measurement of Deoxyribose 3JHH Scalar Couplings Reveals Protein-Binding Induced Changes in the Sugar Puckers of the DNA. Iwahara J, et al. (2001), J. Mag Res. 2001, 153, 262
An efficient NMR experiment for analyzing sugar-puckering in unlabeled DNA:. Couplings via constant time NOESY.
J. Boisbouvier, B. Brutscher, A. Pardi, D. Marion, and J.-P. Simorre (2000), J. Am. Chem. Soc. 122, 6779–6780
NMR determination of sugar-puckers in nucleic acids form CSA-dipolar cross correlated relaxation.
BioNMR in Drug Research 2003 Editor(s): Oliver Zerbe (Wiley-VCH)
Methods for the Measurement of Angle Restraints from Scalar, Dipolar Couplings and from Cross-Correlated Relaxation: Application to Biomacromolecules Chapter 7 p147-178. Christian Griesinger (also for α and ζ) !
O3’
nucleotide unit
α
β
γ
ν4
ν3
δ
ε
O4’
χ
ν0
α and ζ pose problems!
Determinants of 31P chem shift.!
!
ε and ζ correlate. ζ = -317-1.23 ε !
ν1
ν2
ζ
O5’
+ NOE!
+ NOE!
Backbone Experiments:
CT-NOESY, CT-COSY
Bax, A., Tjandra, N., Zhengrong, W., ( 2001). Measurements of 1H-31P dipolar couplings in a DNA
oligonucleotide by constant time NOESY difference spectroscopy, J. Mol. Biol., 19, 367-270.
91.